DNA hybridization: comparison of liquid and solid phase formats.
In nucleic acid hybridization an oligo- or polynucleotide probe is allowed to anneal to its complementary strand which possibly is present in the sample. This offers an extremely specific way to identify and quantify given genes and thus, for instance, given microbes. The annealing reaction is, however, slow since the reactants are present at very low concentrations and the diffusion rate of DNA is slow. To overcome this problem high concentrations of probe are used in order to "drive" the reaction in a pseudo-first order fashion. As a result a positive hybridization is easily masked by the large excess of unreacted probe molecules, unless a powerful fractionation system is used which removes the free probes. A frequently used method is to immobilize the nucleic acids of the sample on a solid support which then after the reaction is easy to wash. The solid support introduces, however, a diffusion barrier which significantly reduces the reaction rate. Thus kinetically solution phase reactions are preferable to solid phase ones. In this communication a test format is described in which the advantages of both solid and solution phase assays are combined. Two probes are used, one carrying a detectable label (the detector probe) and the other an affinity moiety, e.g. biotin (the capture probe). After hybridization in solution a sandwich hybrid is formed in which the target nucleic acid is annealed to the two probes.(ABSTRACT TRUNCATED AT 250 WORDS)
Söderlund
H
H
Orion Corporation, Orion Pharmaceutica, Biotechnology, Helsinki, Finland.
D004247:DNA; D015342:DNA Probes; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction
D015342:DNA Probes; D004247:DNA